The ability to determine the presence of an analyte in a sample is of significant benefit. For example, analytes composed of certain ions and metals, such as those toxic elements belonging to the RCRA-8 metal group (lead (Pb), mercury (Hg), arsenic (As), chromium (Cr), cadmium (Cd), barium (Ba), silver (Ag), and selenium (Se)), pose significant health risks when present in water supplies. It is common to perform sample analysis on drinking water, ground water, and waste water to monitor and safeguard water quality used for human consumption and agricultural purposes, as well as to preserve the environment.
Sample analysis is equally important for medical reasons and for homeland security. Biological fluids, such as blood and those originating from body tissues, also may be tested for a variety of analytes to determine if the body has been exposed to harmful agents or if a disease state exists. In a similar vein, the detection of harmful agents, such as bioterrorist materials (for example, poisons like anthrax), minute quantities of highly explosive materials (for example, C4 plastic explosive and Trinitrotoluene (TNT)), and illegal drug substances and related contraband (for example, cocaine) is important for the safety of both individuals and society at large.
Colorimetric methods are commonly used for the detection of analytes in soil, water, or waste-stream samples, biological samples, body fluids, and the like. In relation to instrument-based methods of analysis, such as atomic absorption spectroscopy, calorimetric methods tend to be rapid and require little in the way of equipment or user sophistication. While conventional calorimetric tests are extremely useful, they only exist for a limited set of analytes, and often cannot detect very small or trace amounts of the analyte.
Recently, colorimetric sensors based upon aptamers have been developed. Aptamers are nucleic acids (such as DNA or RNA) that recognize target effector molecules with high affinity and specificity (Ellington and Szostak 1990, Jayasena 1999). Aptamers have several unique properties that make them an ideal platform for designing highly sensitive and selective analyte sensors. First, in vitro selection methods can be used to obtain aptamers for a wide range of target effector molecules with exceptionally high affinity, having dissociation constants in the picomolar range (Brody and Gold 2000, Jayasena 1999, Wilson and Szostak 1999). Second, aptamers are easier to obtain and less expensive to produce than antibodies, because aptamers can be generated in vitro in short time periods (for example, within days) and at economical cost. Third, aptamers display remarkable structural durability and can be denatured and renatured many times without losing their ability to recognize their targets.
One particularly advantageous calorimetric sensor is an aptamer design that directs assembly or disassembly of metallic particle aggregates in response to an analyte. Metallic particles are exquisitely sensitive calorimetric reagents, having extinction coefficients three orders of magnitude higher than those of organic dyes (Link et al. 1999). Aptamer systems may be designed to bind two or more oligonucleotides that are coupled to particles (oligo-particles), thereby resulting in formation of an aggregate of particles (particle aggregate). Upon exposure to a sample containing the effector molecule (analyte), the aptamer binds to the effector molecule by undergoing a conformational change that precludes or weakens binding of the oligo-particles to each other, and the particle aggregate dissociates. Because particle aggregates display spectral attributes dependent upon the distance between the particles, the aggregation status of the oligo-particles is reflected by the appearance of distinct calorimetric properties. Since aptamers are designed to recognize a specific analyte, the presence of the specific analyte in a sample is reported calorimetrically as the particle aggregates dissociate. An example of this technology is described in U.S. Patent Application Publication No. 20070037171 A1, entitled APTAMER-BASED COLORIMETRIC SENSOR SYSTEMS to Y. Lu et al., published Feb. 15, 2007.
Other types of sensors based upon nucleic acid enzymes (for example, aptazymes, DNAzymes, and RNAzymes) have been described. Nucleic acid enzymes are well known in the art, and have been used in sensor applications designed to detect single analyte species (see, for examples, U.S. Patent Application Publication No. 20030215810 A1, entitled SIMPLE CATALYTIC DNA BIOSENSORS FOR IONS BASED ON COLOR CHANGES to Y. Lu et al., published Nov. 20, 2003; U.S. Patent Application Publication No. 20040175693 A1, entitled NUCLEIC ACID BIOSENSORS to Y. Lu et al., published Sep. 9, 2004).
Because aptamers and nucleic acid enzymes are selected for their ability to bind to specific target effector molecules, colorimetric sensors based on these conventional designs are limited to detecting a single analyte species in a sample. However, there is often a need to detect more than one type of analyte species in a given sample. For example, for a complete environmental analysis of mercury contaminants in a given sample, it is important to analyze the sample for the presence of both organic and inorganic mercury species. Even if aptamer and nucleic acid enzyme-based sensor system designs were available that recognize two or more analyte species, calorimetric sensor designs have not been implemented to permit selective detection of the different analyte species. Thus, sensors capable of simultaneously detecting multiple analytes present in a sample have not been described.